Viruses are self-replicating entities that require either prokaryotic or eukaryotic cells in order to replicate and propagate. When a virus comes in contact with a susceptible cell at first it attaches itself to the cell and then enters it. Once inside the cell, the virus hijacks the protein synthesis machinery of the cell and commands it to manufacture the required components to assemble more copies of itself. After a certain amount of time, the virus copies exit the cell either bursting the cell open or budding through the cell membrane. Viruses are classified according to several taxonomic criteria, one of the most relevant being whether they either have or lack a phospholipid bilayer envelope.
Both enveloped and non-enveloped viruses are found among human and veterinary pathogens. One of the most important veterinary disease caused by a non-enveloped virus is Foot-and-Mouth Disease (FMD) caused by the Foot-and-Mouth Disease Virus (FMDV). Another example of a non-enveloped virus causing big impacts on livestock productivity is Bovine Rotavirus, the causative agent of neonatal diarrhoea in calves. Some examples of enveloped viruses causing veterinary diseases include Bovine Herpesviruses 1 and 5 (BoHV-1 or BHV-1 and BoHV-5 or BHV-5) which are the etiologic agents of Infectious Bovine Rhinotracheitis and Bovine Herpetic Encephalitis respectively; the Bovine Parainfluenza Virus 3 (PI3 o BPIV-3) associated with the bovine respiratory disease (BRD) complex; and the Rabies Virus which is the pathogen causative of lethal encephalitis in both animals and human beings.
FMD is a severe and highly contagious disease affecting cloven-hoofed animals, including cattle, swine, sheep, goats and deer. Foot-and-mouth disease symptoms include fever, blisters in the mouth and on the feet and teats, drop in milk production, loss of appetite and weight and lameness.
The disease is endemic in many parts of the world but is not recognized as a zoonosis. The World Organization for Animal Health (OIE) periodically publishes disease distribution and outbreak maps. The sanitary status granted by the OIE has a profound economic impact in countries with meat trade-dependent economies because of the market restrictions that OIE imposes, especially on countries affected by FMD.
The FMD virus (FMDV) is a non-lipid-enveloped single strand RNA virus featuring an icosahedral symmetry and a described size (diameter) ranging from 28 to 40 nm. The virus belongs to the Aphtovirus genus, within the Picomaviridae family. The whole virus particle is extremely labile in vitro, it dissociates into monomers at temperatures above 56° C. and pH below 6. Seven FMDV serotypes have been reported, designated as O, A, C, SAT1, SAT2, SAT3 and Asia1.
Effective vaccines and stringent control programs have eradicated FMD in most developed countries but, regardless of strict international trade policies, major outbreaks have occurred relatively recently in Europe (2000, 2001) and in Japan (2000, 2010). FMD vaccines are manufactured in plants with NBS biological safety level 4 (OIE). It is estimated that between 2.5 and 3 billion doses are produced annually worldwide.
Rotaviruses are the most common cause of neonatal diarrhea in calves. Rotaviral diarrhea usually affects calves between four days to three weeks old. Calves are usually dull, and reluctant to drink. Over the course of the disease, which can last between 4 to 8 days, there is a significant risk of calf depression, dehydration and secondary infections. The calves will have a decreased appetite and may drool. The death rate can be as high as 50% without intervention.
The economic impact of calf diarrhea on the livestock industry is very substantial and far reaching: in addition to the costs associated with the additional labor, drug expenditures, and calf death, there is the potential economic impact associated with the poor long-term performance of affected calves. It has been found that 80% of calf morbidity from birth to day 21 is due to diarrhea.
Bovine rotavirus is a non-enveloped double-stranded RNA virus that belongs to the family Reoviridae. The virion has an icosahedral shape and features a triple protein capsid structures. The diameter of the virion has been described to be around 80 nanometers.
Vaccination of the dams against rotavirus represents an effective way to fight the disease and to prevent diarrhea in neo-natal calves. Calves should be fed colostrum from dams vaccinated against rotavirus infection 1-3 months before calving.
Infection of cattle by Bovine Herpesviruses can lead to severe diseases. Infectious Bovine Rhinotracheitis (IBR) is a highly contagious, infectious respiratory disease that is caused by Bovine Herpesvirus-1 (BHV-1 or BoHV-1). Bovine Herpesvirus 5 (BHV-5 or BoHV-5) differs from BoHV-1 in its tropism: BoHV-5 is the causative agent of meningoencephalitis in young cattle. BoHV-1 and BoHV-5 are enveloped double-stranded DNA viruses that belong to the Varicellovirus genus and to the Herpesviridae family. They have a spherical to pleomorphic shape and a described diameter of 150-200 nm. The internal protein capsid consists of 162 capsomers and is surrounded by an amorphous tegument.
IBR can affect young and older cattle. In addition to causing respiratory disease, this virus can cause conjunctivitis, abortions, encephalitis, and generalized systemic infections. IBR is characterized by the acute inflammation of the upper respiratory tract. After the first infection, the virus is never fully removed. It stays behind in nerve cells in the brain as a life-long latent (hidden) infection. However, at times of stress the virus can begin to multiply again and may be re-excreted, generally from the nose and the eyes. Therefore an animal which has been infected can never be considered safe. Purchase of infected animals is the main source of new infections. Diseases caused by the virus can be serious; therefore it is a barrier to international trade.
Since BHV-1 is a ubiquitous, highly contagious virus, vaccination is recommended as soon as passive immunity in calves has disappeared, usually around four to six months of age. Currently available vaccines for IBR include modified-live-virus (MLV) vaccines and inactivated or killed-virus (KV) vaccines.
Bovine Parainfluenza-3 (PI3) is a highly contagious respiratory disease prevalent in the cattle population. The disease is associated with the bovine respiratory disease complex (BRD).
Infection by PI3 virus (PIV-3) alone causes only mild diseases. The clinical signs include fever, cough, watery nasal and lacrimal discharge as well as increased rate of respiration and an increase breathing sounds. Infection can increase morbidity of other viral diseases such as bovine viral diarrhea and infectious bovine rhinotracheitis. The impact of PI3 infection is more significant when coupled with secondary bacterial pneumonia.
Parainfluenza-3 virus (PIV-3) is a negative-stranded RNA enveloped virus belonging to the Respirovirus genus within the Paramyxoviridae family. The virus has a spherical shape and a described diameter of about 150 nm.
Prevention through vaccination is the best way to prevent any large impact of Parainfluenza-3. Most PI3 vaccines are combined with other respiratory viruses like for example bovine herpesvirus-1 (BHV-1). The vaccine is available in modified live or inactivated form.
Rabies is a zoonotic disease transmitted from animals to humans, and caused by the rabies virus, of the Lyssavirus genus within the family Rhabdoviridae. The rabies virus is a negative-stranded RNA enveloped virus with a bullet like shape of about 180 nm in length and a cross-sectional diameter of about 75 nm.
All mammals are thought to be susceptible to the rabies virus. Domestic dogs are the most common reservoir of the virus, with more than 95% of human deaths caused by dog-mediated rabies.
The paralytic clinical manifestation of rabies is the most common form in cattle, pigs and horses. A bite from an infected wild animal, such as a fox or raccoon, is a common method of infection in cattle although the disease is also transmitted by hematophagous bats, a.k.a. “vampire” bats in tropical and subtropical areas. It is a serious threat to animal production with several thousand head of cattle losses each year and a human health concern because of contact between humans and rabid cattle during normal rearing operations.
With the exception of Antarctica, rabies is endemic in all continents. Of the tens of thousands of deaths occurring annually due to rabies, 95% of cases are reported in Asia and Africa.
Rabies is a 100% vaccine-preventable disease: safe and effective inactivated virus vaccines are available for dogs, cattle and humans. Post-exposure prophylactic vaccinations are required for treating individuals who have had no previous immunization.
Over the last decades, human and veterinarian health industries have witnessed increasingly stringent government regulations related to Good Manufacturing Practice (GMP) for the production of pharmaceutical and biopharmaceuticals products. New quality concepts have arisen, like Process Analytical Technology (PAT), which Food and Drug Administration (FDA) defines as a mechanism to design, analyze, and control pharmaceutical manufacturing processes through the measurement of Critical Process Parameters (CPP) that affect Critical Quality Attributes (CQA). PAT emphasizes the importance of controlling the production process as a means to achieve the highest quality standard for the final product. PAT represents an especially useful tool for the quality control of intermediate and final products in the vaccine industry. Indeed, the efficacy of inactivated virus vaccines, which are routinely used as part of eradication programs and in emergency contexts, is highly dependent on the antigenic payload formulated in each dose and on virus integrity.
As of today, the in-process quality controls of whole virus antigens in intermediate process streams relies mainly on the performance of titration assays when the virus is still alive (before the inactivation step) or on immunochemical methods like ELISA (Enzyme-Linked ImmunoSorbent Assay) or single radial immunodiffusion (SRID) tests and in some cases real-time quantitative PCR when the virus has already been inactivated.
ELISA detection kits are commonly used for the detection of disease-causative agents in body fluids, both for humans and animals. ELISA kits are generally designed to detect the antibody response to the infectious agents but not the infectious agents themselves. Although ELISA detection assays are used in some cases for the monitoring and quality control of viral antigens during manufacturing process, these assays have to be developed on a case by case basis and cannot be standardized to different kind of viruses as the conditions of the assay heavily relies on the detection reagents used in the kits, like monoclonal or polyclonal antibodies. The same is true for SRID assays.
Moreover, ELISA methods can yield only a rough quantification of virus particles based on the colorimetric titration of an antibody-antigen reaction. The results are greatly influenced by the dilution factor of the sample and the sample matrix. Consequently, the ELISA assay has to be adapted to the different kind of samples of the different manufacturing steps and cannot be considered as a useful tool for Process Analytical Technology (PAT).
In addition, ELISA assays or quantitative PCR methods do not yield any information on the size nor structural integrity of the virus particles, as both methods cannot discriminate between whole or ruptured viral antigens. Knowing the size and structural integrity of the virus particles are important to ensure the immunizing efficacy of vaccines.
For the FMD virus, the 140S (146S) quantitative sucrose density gradient analysis, as developed by Barteling and Meloen (Barteling S J, Meloen R H. A simple method for the quantification of 140S particles of foot-and-mouth disease virus (FMDV). Arch Gesamte Virusforsch, 1974; 45(4):362-4), is the recommended method to quantify virus antigen and, on that basis, formulate vaccines. The principle of the 146S method is to separate the FMD viral particles based on their sedimentation coefficient in the sucrose gradient. Therefore, the technique provides only an indirect measurement of the integrity of the viral particles.
A number of international efforts have been attempted in order to standardize the method but there is as yet neither a harmonized protocol nor an international FMDV standard. The technical complexities of the method and the requirement of specialized items of equipment have probably contributed to this situation (Barteling S J. Need for further standardization of the 146—S test as the basis for final—Foot-and-Mouth Disease (FMD) Vaccine formulation. EUFMD Research Group Meeting 1999—Appendix 17).
The lack of a standardized method to measure the active ingredient present in viral vaccines is an important factor to explain why expensive and cumbersome clinical trials, typically involving large animals (the target species) or laboratory animals are still required for registration of viral vaccines and batch release.
For example in the case of the Rabies vaccine, the NIH potency test has been used over several decades as the reference assay for potency testing of vaccine batches. This cruel and highly complicated in-vive assay requires the use of 150 mice per batch and includes the intracranial challenge of the mice with a pathogenic rabies strain.
Current international efforts working on the harmonization of methods for potency controls of vaccine and biopharmaceuticals molecules are focusing on the 3R principle: Reduction of the number of animals used; Replacement of animals with alternative technique, and Refinement of the ways experiments are carried out to make sure animals suffer as little as possible.
For the vaccine industry and especially for vaccines based on full-size antigens (like inactivated viral vaccines or live attenuated viral vaccines), methods and equipment capable of reliably quantifying and determining the size of particles in the nanometer (nm) range would represent important tools for the implementation of in-process controls meant to ensure the quality of the viral antigens produced at every step of the process. Nevertheless, the analysis of complex process streams containing virus particles is a much more difficult task than the analysis of common recombinant proteins like monoclonal antibodies (mAbs). One challenge is that manufacturing practices for many current vaccines were developed decades ago, before the rise of serum-free media culture technology. The serum containing medium used in many manufacturing processes contains a very high content of proteins. Also, the yield of virus particles obtained from the infection of cell culture is generally in the order of milligram per liters, which is several orders of magnitude lower compared to the expression of recombinant proteins such as mAbs. Also of great importance is the fact that some viruses have lytic replication cycle that triggers the destruction of the cell. The release in the cell culture medium of all the materials contained in the cytoplasm and the nucleus of the cells, including all the genomic material and lipid residues from the cell membrane will make the analysis more difficult.
Since size exclusion chromatography separates different molecules based on their hydrodynamic volumes, molecules of different molecular weights but similar hydrodynamic volumes are prone to show very similar mobility behavior during chromatography runs. In the conditions of very complex process streams, size exclusion chromatography is usually unable to provide a resolution good enough in order to separate the peak of virus particles of interest from other process contaminants of similar hydrodynamic volume such as large molecules of DNA and large lipid residues.
Protein or viral-based vaccines are vaccine models usually employed for delivering antigens to a subject for immunization. It is extremely crucial that the antigen being delivered is of its correct or native conformation such that appropriate epitopes can be presented to induce specific and effective immune responses. Therefore, misfolded, degraded or aggregated antigen may have reduced efficacy in, or even not be capable of, triggering specific immune responses. Dynamic Light Scattering (DLS) helps to assess the efficacy of protein or viral-based vaccines in terms of their integrity and stability. By analyzing the DLS profile, one would be able to check whether the antigens have been degraded or aggregated over time or upon storage in a particular buffer or temperature, and thereby assuring the efficacy of the vaccines.
Separation methodologies such as size-exclusion chromatography, when used alone, usually give rough estimates of particle size by close scrutiny of mobility behavior but these assessments are not sensitive enough to detect subtle modifications of antigen dimension. In contrast, DLS sensitively and accurately detects and estimates size of particles, and hence allows monitoring subtle differences of the particles' size. However, DLS can only analyze the integrity and estimate the size of particles with high purity.
Due to the economic relevance of these livestock diseases worldwide, there is a continuous need of improvements in the prevention tools, particularly regarding improved vaccines and/or methods of preparation thereof. The present invention provides methods for the quantification and characterization of viruses, including the enveloped and non-enveloped viruses, and the RNA and DNA-based viruses, and related products in a high-throughput and accurate manner. With the growing demand for both quantity and quality of veterinary vaccines, the present invention is a very useful and cost-effective tool for the quality control of the viral vaccines.
As an example and in the case of Foot-and-Mouth Disease, the world biggest vaccine market, the Chinese market, requires 1.7 billion doses (of 2 milliliters each) per year. Considering that one average industrial batch of vaccine represents 5 million doses (i.e., a 10000 liters batch), this means that around 340 batches of FMD vaccines are produced each year in China by different manufacturers. As of today and because of this huge number of batches produced, the Chinese Veterinary Regulatory Authority has forfeited the responsibility of controlling the quality of the FMD vaccine batches and has to rely on the performance of quality control tests by each vaccine manufacturer. The quality control of FMD vaccines is usually monitored using in vivo potency testing which allows assessment of the quality of the vaccines in bulk quantity but requires complex and cumbersome procedures. Currently, there are no other in vitro techniques available that would enable the quality control of such a great quantity of batches of vaccine in a timely manner.
The present invention, for the first time, introduces an in vitro system that is capable of quantifying the antigen payload per dose and characterizing the size and the integrity of the viral antigen in each vaccine batch, in a high throughput manner, no matter if the antigen is a live-attenuated or inactivated virus. The present invention implements a much more simplified system than the in vivo potency testing system for the quality control of viral vaccines and thereby improves the quality of the products. It is estimated that the present invention can monitor the quality of 340 batches of vaccines in just a few days.
Thus, the present invention would eventually guarantee a better quality of the vaccines and confer a better protection against veterinary diseases to the animal and human population.